Sylvain Marthey

Assessing the Performance of Single-Copy Genes for Recovering Robust Phylogenies

Phylogenies involving nonmodel species are based on a few genes, mostly chosen following historical or practical criteria. Because gene trees are sometimes incongruent with species trees, the resulting phylogenies may not accurately reflect the evolutionary relationships among species. The increase in availability of genome sequences now provides large numbers of genes that could be used for building phylogenies. However, for practical reasons only a few genes can be sequenced for a wide range of species. Here we asked whether we can identify a few genes, among the single-copy genes common to most fungal genomes, that are sufficient for recovering accurate and well-supported phylogenies. Fungi represent a model group for phylogenomics because many complete fungal genomes are available. An automated procedure was developed to extract single-copy orthologous genes from complete fungal genomes using a Markov Clustering Algorithm (Tribe-MCL). Using 21 complete, publicly available fungal genomes with reliable protein predictions, 246 single-copy orthologous gene clusters were identified. We inferred the maximum likelihood trees using the individual orthologous sequences and constructed a reference tree from concatenated protein alignments. The topologies of the individual gene trees were compared to that of the reference tree using three different methods. The performance of individual genes in recovering the reference tree was highly variable. Gene size and the number of variable sites were highly correlated and significantly affected the performance of the genes, but the average substitution rate did not. Two genes recovered exactly the same topology as the reference tree, and when concatenated provided high bootstrap values. The genes typically used for fungal phylogenies did not perform well, which suggests that current fungal phylogenies based on these genes may not accurately reflect the evolutionary relationships among species. Analyses on subsets of species showed that the phylogenetic performance did not seem to depend strongly on the sample. We expect that the best-performing genes identified here will be very useful for phylogenetic studies of fungi, at least at a large taxonomic scale. Furthermore, we compare the method developed here for finding genes for building robust phylogenies with previous ones and we advocate that our method could be applied to other groups of organisms when more complete genomes are available.

FUNYBASE: a FUNgal phYlogenomic dataBASE

BackgroundThe increasing availability of fungal genome sequences provides large numbers of proteins for evolutionary and phylogenetic analyses. However the heterogeneity of data, including the quality of genome annotation and the difficulty of retrieving true orthologs, makes such investigations challenging. The aim of this study was to provide a reliable and integrated resource of orthologous gene families to perform comparative and phylogenetic analyses in fungi.DescriptionFUNYBASE is a database dedicated to the analysis of fungal single-copy genes extracted from available fungal genomes sequences, their classification into reliable clusters of orthologs, and the assessment of their informative value for phylogenetic reconstruction based on amino acid sequences. The current release of FUNYBASE contains two types of protein data: (i) a complete set of protein sequences extracted from 30 public fungal genomes and classified into clusters of orthologs using a robust automated procedure, and (ii) a subset of 246 reliable ortholog clusters present as single copy genes in 21 fungal genomes. For each of these 246 ortholog clusters, phylogenetic trees were reconstructed based on their amino acid sequences. To assess the informative value of each ortholog cluster, each was compared to a reference species tree constructed using a concatenation of roughly half of the 246 sequences that are best approximated by the WAG evolutionary model. The orthologs were classified according to a topological score, which measures their ability to recover the same topology as the reference species tree. The full results of these analyses are available on-line with a user-friendly interface that allows for searches to be performed by species name, the ortholog cluster, various keywords, or using the BLAST algorithm. Examples of fruitful utilization of FUNYBASE for investigation of fungal phylogenetics are also presented.ConclusionFUNYBASE constitutes a novel and useful resource for two types of analyses: (i) comparative studies can be greatly facilitated by reliable clusters of orthologs across sets of user-defined fungal genomes, and (ii) phylogenetic reconstruction can be improved by identifying genes with the highest informative value at the desired taxonomic level.

Comparative Genome Analyses Reveal Distinct Structure in the Saltwater Crocodile MHC

The major histocompatibility complex (MHC) is a dynamic genome region with an essential role in the adaptive immunity of vertebrates, especially antigen presentation. The MHC is generally divided into subregions (classes I, II and III) containing genes of similar function across species, but with different gene number and organisation. Crocodylia (crocodilians) are widely distributed and represent an evolutionary distinct group among higher vertebrates, but the genomic organisation of MHC within this lineage has been largely unexplored. Here, we studied the MHC region of the saltwater crocodile (Crocodylus porosus) and compared it with that of other taxa. We characterised genomic clusters encompassing MHC class I and class II genes in the saltwater crocodile based on sequencing of bacterial artificial chromosomes. Six gene clusters spanning ∼452 kb were identified to contain nine MHC class I genes, six MHC class II genes, three TAP genes, and a TRIM gene. These MHC class I and class II genes were in separate scaffold regions and were greater in length (2–6 times longer) than their counterparts in well-studied fowl B loci, suggesting that the compaction of avian MHC occurred after the crocodilian-avian split. Comparative analyses between the saltwater crocodile MHC and that from the alligator and gharial showed large syntenic areas (>80% identity) with similar gene order. Comparisons with other vertebrates showed that the saltwater crocodile had MHC class I genes located along with TAP, consistent with birds studied. Linkage between MHC class I and TRIM39 observed in the saltwater crocodile resembled MHC in eutherians compared, but absent in avian MHC, suggesting that the saltwater crocodile MHC appears to have gene organisation intermediate between these two lineages. These observations suggest that the structure of the saltwater crocodile MHC, and other crocodilians, can help determine the MHC that was present in the ancestors of archosaurs.

Expressed sequences tags of the anther smut fungus, Microbotryum violaceum, identify mating and pathogenicity genes

BackgroundThe basidiomycete fungus Microbotryum violaceum is responsible for the anther-smut disease in many plants of the Caryophyllaceae family and is a model in genetics and evolutionary biology. Infection is initiated by dikaryotic hyphae produced after the conjugation of two haploid sporidia of opposite mating type. This study describes M. violaceum ESTs corresponding to nuclear genes expressed during conjugation and early hyphal production.ResultsA normalized cDNA library generated 24,128 sequences, which were assembled into 7,765 unique genes; 25.2% of them displayed significant similarity to annotated proteins from other organisms, 74.3% a weak similarity to the same set of known proteins, and 0.5% were orphans. We identified putative pheromone receptors and genes that in other fungi are involved in the mating process. We also identified many sequences similar to genes known to be involved in pathogenicity in other fungi. The M. violaceum EST database, MICROBASE, is available on the Web and provides access to the sequences, assembled contigs, annotations and programs to compare similarities against MICROBASE.ConclusionThis study provides a basis for cloning the mating type locus, for further investigation of pathogenicity genes in the anther smut fungi, and for comparative genomics.

Identification of differentially expressed genes in chickens differing in muscle glycogen content and meat quality

BackgroundThe processing ability of poultry meat is highly related to its ultimate pH, the latter being mainly determined by the amount of glycogen in the muscle at death. The genetic determinism of glycogen and related meat quality traits has been established in the chicken but the molecular mechanisms involved in variations in these traits remain to be fully described. In this study, Chicken Genome Arrays (20 K) were used to compare muscle gene expression profiles of chickens from Fat (F) and Lean (L) lines that exhibited high and low muscle glycogen content, respectively, and of individuals exhibiting extremely high (G+) or low (G-) muscle glycogen content originating from the F2 cross between the Fat and Lean lines. Real-time RT-PCR was subsequently performed to validate the differential expression of genes either selected from the microarray analysis or whose function in regulating glycogen metabolism was well known.ResultsAmong the genes found to be expressed in chicken P. major muscle, 197 and 254 transcripts appeared to be differentially expressed on microarrays for the F vs. L and the G+ vs. G- comparisons, respectively. Some involved particularly in lipid and carbohydrate metabolism were selected for further validation studies by real-time RT-PCR. We confirmed that, as in mammals, the down-regulation of CEBPB and RGS2 coincides with a decrease in peripheral adiposity in the chicken, but these genes are also suggested to affect muscle glycogen turnover through their role in the cAMP-dependent signalling pathway. Several other genes were suggested to have roles in the regulation of glycogen storage in chicken muscle. PDK4 may act as a glycogen sensor in muscle, UGDH may compete for glycogen synthesis by using UDP-glucose for glucoronidation, and PRKAB1, PRKAG2, and PHKD may impact on glycogen turnover in muscle, through AMP-activated signalling pathways.ConclusionsThis study is the first stage in the understanding of molecular mechanisms underlying variations in poultry meat quality. Large scale analyses are now required to validate the role of the genes identified and ultimately to find molecular markers that can be used for selection or to optimize rearing practices.

Genes under positive selection in a model plant pathogenic fungus, Botrytis

The rapid evolution of particular genes is essential for the adaptation of pathogens to new hosts and new environments. Powerful methods have been developed for detecting targets of selection in the genome. Here we used divergence data to compare genes among four closely related fungal pathogens adapted to different hosts to elucidate the functions putatively involved in adaptive processes. For this goal, ESTs were sequenced in the specialist fungal pathogens Botrytis tulipae and Botrytis ficariarum, and compared with genome sequences of Botrytis cinerea and Sclerotinia sclerotiorum, responsible for diseases on over 200 plant species. A maximum likelihood-based analysis of 642 predicted orthologs detected 21 genes showing footprints of positive selection. These results were validated by resequencing nine of these genes in additional Botrytis species, showing they have also been rapidly evolving in other related species. Twenty of the 21 genes had not previously been identified as pathogenicity factors in B. cinerea, but some had functions related to plant-fungus interactions. The putative functions were involved in respiratory and energy metabolism, protein and RNA metabolism, signal transduction or virulence, similarly to what was detected in previous studies using the same approach in other pathogens. Mutants of B. cinerea were generated for four of these genes as a first attempt to elucidate their functions.

Finding candidate genes under positive selection in Non‐model species: examples of genes involved in host specialization in pathogens

Numerous genes in diverse organisms have been shown to be under positive selection, especially genes involved in reproduction, adaptation to contrasting environments, hybrid inviability, and host‐pathogen interactions. Looking for genes under positive selection in pathogens has been a priority in efforts to investigate coevolution dynamics and to develop vaccines or drugs. To elucidate the functions involved in host specialization, here we aimed at identifying candidate sequences that could have evolved under positive selection among closely related pathogens specialized on different hosts. For this goal, we sequenced c. 17 000–32 000 ESTs from each of four Microbotryum species, which are fungal pathogens responsible for anther smut disease on host plants in the Caryophyllaceae. Forty‐two of the 372 predicted orthologous genes showed significant signal of positive selection, which represents a good number of candidate genes for further investigation. Sequencing 16 of these genes in 9 additional Microbotryum species confirmed that they have indeed been rapidly evolving in the pathogen species specialized on different hosts. The genes showing significant signals of positive selection were putatively involved in nutrient uptake from the host, secondary metabolite synthesis and secretion, respiration under stressful conditions and stress response, hyphal growth and differentiation, and regulation of expression by other genes. Many of these genes had transmembrane domains and may therefore also be involved in pathogen recognition by the host. Our approach thus revealed fruitful and should be feasible for many non‐model organisms for which candidate genes for diversifying selection are needed.

Characterisation and Comparison of Lactating Mouse and Bovine Mammary Gland miRNomes

Background The mammary gland is a dynamic organ that undergoes important physiological changes during reproductive cycles. Until now, data regarding the characterisation of miRNA in the mammary gland have been scarce and mainly focused on their abnormal expression in breast cancer. Our goal was to characterise the microRNA (miRNA) involved in mechanisms regulating the mammary function, with particular focus on the lactation stage. Methodology/principal findings Using high-throughput sequencing technology, the exhaustive repertoires of miRNA expressed (miRNome) in mouse and bovine mammary glands during established lactation were identified, characterized and compared. Furthermore, in order to obtain more information on miRNA loading in the RNA-induced silencing complex (RISC), the miRNome was compared with that obtained from RNA associated with the AGO2 protein (AGO2-miRNome) in mouse lactating mammary gland. This study enabled the identification of 164 and 167 miRNA in mouse and bovine, respectively. Among the 30 miRNA most highly expressed in each species, 24 were common to both species and six of them were preferentially highly expressed in lactating than non-lactating mammary gland. The potential functional roles of these 24 miRNA were deduced using DIANA-miRPath software, based on miRNA/mRNA interactions. Moreover, seven putative novel miRNA were identified. Using DAVID analysis, it was concluded that the predicted targets of two of these putative novel miRNA are involved in mammary gland morphogenesis. Conclusion/significance Our study provides an overview of the characteristics of lactating mouse and bovine mammary gland miRNA expression profiles. Moreover, species-conserved miRNA involved in this fundamental biological function were identified. These miRNomes will now be used as references for further studies during which the impact of animal breeding on the miRNA expression will be analysed.

Transcriptomic Analysis Brings New Insight into the Biological Role of the Prion Protein during Mouse Embryogenesis

The biological function of the Prion protein remains largely unknown but recent data revealed its implication in early zebrafish and mammalian embryogenesis. To gain further insight into its biological function, comparative transcriptomic analysis between FVB/N and FVB/N Prnp knockout mice was performed at early embryonic stages. RNAseq analysis revealed the differential expression of 73 and 263 genes at E6.5 and E7.5, respectively. The related metabolic pathways identified in this analysis partially overlap with those described in PrP1 and PrP2 knockdown zebrafish embryos and prion-infected mammalian brains and emphasize a potentially important role for the PrP family genes in early developmental processes.

Prion Protein and Shadoo Are Involved in Overlapping Embryonic Pathways and Trophoblastic Development

The potential requirement of either the Prion or Shadoo protein for early mouse embryogenesis was recently suggested. However, the current data did not allow to precise the developmental process that was affected in the absence of both proteins and that led to the observed early lethal phenotype. In the present study, using various Prnp transgenic mouse lines and lentiviral vectors expressing shRNAs that target the Shadoo-encoding mRNA, we further demonstrate the specific requirement of at least one of these two PrP-related proteins at early developmental stages. Histological analysis reveals developmental defect of the ectoplacental cone and important hemorrhage surrounding the Prnp-knockout-Sprn-knockdown E7.5 embryos. By restricting the RNA interference to the trophoblastic cell lineages, the observed lethal phenotype could be attributed to the sole role of these proteins in this trophectoderm-derived compartment. RNAseq analysis performed on early embryos of various Prnp and Sprn genotypes indicated that the simultaneous down-regulation of these two proteins affects cell-adhesion and inflammatory pathways as well as the expression of ectoplacental-specific genes. Overall, our data provide biological clues in favor of a crucial and complementary embryonic role of the prion protein family in Eutherians and emphasizes the need to further evaluate its implication in normal and pathological human placenta biology.